Half of all triple negative breast cancer (TNBC) patients harbor significant residual cancer burden following standard neoadjuvant chemotherapy treatment, resulting in distant metastasis and death for most of these patients. Clinically overt metastases are incurable, so there is an urgent need to develop targeting strategies to eradicate metastatic cells growing in secondary organs. Intra-tumor heterogeneity (ITH) is pervasive in TNBC as is a barrier to development of effective therapeutic strategies. Dr. Echeverria?s proposal aims to unravel this complexity, with the long-term goal of distilling evolutionary patterns that can be leveraged therapeutically to halt local and distant disease progression. Dr. Echeverria?s postdoctoral studies elucidated patterns of clonal dynamics in TNBC chemoresistance and metastasis using patient-derived xenograft (PDX) models and found that multi-organ metastases are seeded and predominated by the same subclone populations. Furthermore, she identified mitochondrial oxidative phosphorylation (OXPHOS) as a functional vulnerability in PDX models and demonstrated efficacy of a novel OXPHOS inhibitor (OXPHOSi) in chemoresistant TNBCs. This proposal aims to address the following hypotheses: that transcriptomic ITH is present in chemoresistant TNBCs and multi- organ metastases, that subclones in multi-organ metastases share transcriptomic features, that TNBC subclones modulate metabolic programs as they resist chemotherapy and metastasize, and that OXPHOS is a functional vulnerability of multi-organ metastases in TNBC. These hypotheses will be tested in three specific aims. In Aim 1, the subclonal dynamics of transcriptomic programs as cells resist NACT and OXPHOSi treatment will be elucidated by single-cell RNA sequencing (SCRS) and functional metabolic assays. Aim 2 will delineate transcriptomic ITH in the most common sites of human TNBC metastasis by conducting SCRS on lung, liver, and brain metastases isolated from PDXs. These studies will be complemented by functional metabolic experiments and analyses of tumor biopsies obtained from TNBC patients. In Aim 3, the functional role of OXPHOS in driving multi-organ metastasis will be determined by genetic and pharmacologic disruption of OXPHOS activity in PDX models and TNBC cell lines. These studies will elucidate functions of candidate metastasis driver genes discovered in Dr. Echeverria?s postdoctoral studies and will proceed with additional candidate drivers discovered by SCRS in this proposal. Dr. Echeverria has assembled an outstanding team of collaborators and mentors that will enable her to apply novel technologies to understand TNBC chemoresistance and metastasis and will facilitate her development as an independent investigator. The immersive research environment at MD Anderson has provided Dr. Echeverria with rigorous inter-disciplinary training in cancer research and she will establish her independent research program at a research university where she can continue her basic and translational breast cancer research.